Use of an agarose gel slab which may be dried and rehydrated in an aqueous separation solvent. The rehydrated gel slab has a porosity substantially similar to that of the initially produced gel slab and is in a state of equilibration with the separation solvent and ready for use in various systems such as those for separation of biological molecules.
Agarose is a purified linear galactan hydrocolloid isolated from agar or recovered directly from agar-bearing marine algae (The Agarose Monograph (1982) FMC Corporation, Rockland, Maine). Although agarose was initially thought to be the neutral agarobiose fraction of agar, it is known to sometimes contain charged groups such as sulfate ester, ketal pyruvate, and carboxyl groups (The Agarose Monograph (1982) FMC Corporation, Rockland, Maine). The hydrated counter-ions associated with these anionic residues are responsible for the electroendosmosis observed in immunoelectrophoresis, but these charged groups can be removed or blocked to permit the use of agarose in isoelectric focusing.
Agarose is a successful anticonvection medium because it exhibits high gel strength at low concentrations. This gel strength is the result of a three-dimensional gel network based on hydrogen bonding and hydrophobic interaction. Agarose gels are used in a variety of applications and forms a macroporous, nonrestrictive matrix which allows rapid diffusion of high molecular weight macro-molecules. A large pore size, shorter run times, and lack of toxicity are the major advantages of choosing agarose over polyacrylamide for the analysis of macro-molecules. If compounds such as urea that disrupt hydrogen bond formation are present during the casting and formation of the gel, gel strength will be decreased (The Agarose Monograph (1982) FMC Corporation, Rockland, Me.) or the gel may not even form. Previously, these compounds could only be incorporated into preformed agarose gels by extended dialysis, which was not only time consuming, but was expensive with compounds such as the ampholytes necessary for isoelectric focusing.
Although it has been reported that agarose gels can be formed in the presence of 8 M urea (Olsson, I., and Laas, T. (1981) J. Chromatography 215, 373-378), these gels had a significantly lower gel strength than normal gels and could not be readily blotted with nitrocellulose for transfer of separated molecules. In addition, since these gels must cure overnight at 21.degree. C., a significant amount of cyanate forms in the gel (Dirnhuber, P., and Schutz, F. (1948) Biochem. J., 42:628-632), which should be removed before the analysis of proteins.
The low gel strength associated with gels formed in the presence of a high urea concentration has been postulated to be related to a double helix structure of agarose which is stabilized by hydrogen bonding and hydrophobic interactions ((The Agarose Monograph (1982) FMC Corporation, Rockland, Maine). Compounds such as urea that disrupt hydrogen bonding tend to disrupt the agarose structure. In contrast, the structure of polyacrylamide is based on covalent bonding, and reagents that disrupt hydrogen bonding are compatible with the polyacrylamide gel matrix. However, the generally smaller pore size in polyacrylamide gel as compared to agarose gel, detracts from its use with large biological molecules.
In the processes of the present invention, over 4 M and up to 10 M urea may be absorbed directly into the dried agarose gel in 30 minutes. This method of rehydration and equilibration does not cause a noticeable decrease in gel strength and minimizes final cyanate concentrations. In addition, many other substances that would normally be inactivated or modified by the high temperatures preceding gelation of the agarose can be rapidly incorporated directly into the rehydratable agarose gels at 4.degree. C. The rehydratable agarose gels can be used with any procedure employing a slab or film of agarose, including isoelectric focusing, immunoelectrophoresis and Ouchterlony immunodiffusion analysis.
Rehydratable agarose gels have previously been described, for example in Renn et al., (1970) U.S. Pat. No. 3,527,712 and Boschetti et al., (1977) U.S. Pat. No. 4,048,377. In these patents, either hydrocolloids or linear polyacrylamides (LPA) were incorporated into the agarose gels during gel formation. These compounds allowed the gels to be dehydrated and stored in a dry state. Unfortunately, the size, characteristics, and amount of the hydrocolloids and LPA used in these two patents necessitated their removal after rehydration of the agarose gels. Therefore, although the agarose gels could be rehydrated from a dry state, the subsequently required water dialysis to remove the hydrocolloids or LPA from the gels resulted in gels that still had to be dialyzed for equilibration with the reagents of choice. As a result, these rehydratable gels offered no advantage over regular gels for the rapid incorporation of 8 M urea or other reagents, into the gel matrix.
A dried rehydratable film containing agarose and linear acrylamide or methacrylamide was described in U.S. Pat. No. 4,048,377. This reference refers to a gel comprising, at most, 5% agarose and 3 to 6% linear polymer such as polyacrylamide. This gel was dried and stored for periods of time up to 8 months at 4.degree. C. The dried gel film could be rehydrated in aqueous buffers for use in separation procedures. At this time of dried gel rehydration, this reference indicated that the linear polymer, upon the several hours of rehydration, was removed from the gel. The maintenance of gel porosity was not addressed in this reference since it was assumed that all linear polymer was removed to recreate the original gel. The present invention involves a unique methodology procedure for rapidly preparing an equilibrated gel with substantially the original porosity.
U.S. Pat. No. 3,922,432, issued to Rann, describes a thin layer gel medium for use in diffusion or affinity separation procedures. A particulate material, which may be of various substances such as polyacrylamide, carbohydrates or other water-sorptive matter, was attached to a backing sheet and could be hydrated by immersion in water and then utilized for separative purposes.
U.S. Pat. No. 3,878,100, issued to Bixler, describes a thin layer separation medium for use in molecular diffusion or other separation procedures. A thin slab comprising substances such as agarose, sodium alginate and polyethylene glycol may be prepared, dried and then rehydrated for use.
U.S. Pat. No. 3,578,604, issued to Ureal describes a gel comprising agarose and 3 to 7% cross-linked polyacrylamide. The resultant gels, in one example, were cast, washed and dehydrated in a 37.degree. C. oven for 16 to 24 hours. The dehydrated gels were rehydrated by immersion in an aqueous bath for 8 to 16 hours.
U.S. Pat. No. 3,527,712, issued to Renn, describes gels comprising agarose and macro-molecular hydrocolloids present at a concentration in the gel between about 0.1% and about 1.0%. The gel is converted into particles, dried and then rehydrated for usage. The agarose-hydrocolloid mixture may also be formed as a thin film which may be dried and subsequently rehydrated and having the hydrocolloid removed by soaking in water.
U.S. Pat. No. 3,875,044, issued to Renn et al., describes a three layer bonded assembly including a hydratable gel material surrounding by water impervious layers.
U.S. Pat. No. 4,290,911, issued to Cook et al., describes a finely divided solid blend purified agarose and a nonionic gum which is a suitable medium for separation techniques such as isoelectric focusing.
U.S. Pat. No. 4,319,976, issued to Gurske, describes an electrophoretic gel comprising polysaccharide such as agarose and an acid polysaccharide comprising carboxyl groups.
Andrews (page 148-153, 1986) ELECTROPHORESIS, Oxford Press) describes composite polyacrylamide-agarose gels for use in the separation of biological molecules.
Horowitz et al. (Anal. Biochem., 143:333-340 (1984)) describe the electrophoresis of proteins and nucleic acids on acrylamide-agarose gels without covalent cross-linking. No particular advantages of this system appear to be outstanding, according to this reference.
Bode (Anal. Biochem., 83:204-210 (1977)) describes the use of mixed gels comprising agar-agar and liquid polyacrylamide and studies their molecular sieving properties.
Johansson et al. (Anal. Biochem., 59:200-213 (1974)) describe a method for isoelectric focusing of proteins in gels made from a mixture of purified agarose and non-cross linked polyacrylamide. It was found in this reference that polyacrylamide reduced the endoosmotic effects of agarose and sharpened protein bands obtained by separations on the gel.
Bode (Anal. Biochem., 83:364-371 (1977)) describes molecular sieving effects as influenced by the length of single polymer chains, more particularly linear polyacrylamide.
Abbreviations used herein include IEF, isoelectric focusing; LPA, linear polyacrylamide; NFDM, non-fat dried milk; PAP, peroxidase anti-peroxidase; TBS, tris-buffered saline; and ug, microgram.